Project Summary Osteoarthritis (OA) is the most common joint disease, and currently there is no effective means of preventing or slowing joint degeneration. However, growth factors and cytokines are strongly implicated in initiating and aggravating OA lesions. Thus, a molecular understanding of the interplay among these molecules will provide invaluable information toward the search for novel therapeutic targets for OA. Our genome-wide screen for dif- ferentially expressed genes in OA led to the isolation of progranulin (PGRN) as an OA-associated growth fac- tor, and genetic screen for PGRN binding partners led to the isolation of TNFR as the PGRN-binding receptor (Tang, Science, 2011). We have developed an engineered protein named Atsttrin which is composed of three TNFR-binding domains of PGRN, and Atsttrin surpassed PGRN in treating inflammatory arthritis. During the initial funding period, we have successfully identified 14-3-3?, an important intracellular signaling molecule, as a novel component of TNFR2 complexes in response to PGRN stimulation in a proteomics screen. In addition, knockout of 14-3-3? in chondrocytes abolished PGRN's signaling. Further, we recently isolated DR3, the high- est homology to TNFR1, as a novel additional TNFR member that binds to PGRN/Atsttrin, and PGRN/Atsttrin disturbed the interaction between DR3 and TNF-like ligand 1A (TL1A). Thus, this competitive continued appli- cation is primarily based on 1) the identification of 14-3-3? as a novel component of PGRN/TNFR2 pathway, and 2) the isolation of DR3 as a novel additional PGRN-binding receptor. The central hypothesis of this renew- al is that PGRN exerts its chondroprotective role in the pathogenesis of OA through its a) recruitment of 14-3-3? to TNFR2 and activation of the PGRN/TNFR2 anabolic pathway; and b) interplay with and inhibi- tion of TNF?/TNFR1 and TL1A/DR3 inflammatory/catabolic pathways. The Specific Aims are: (1) What are the molecular mechanisms and signaling pathways by which PGRN and Atsttrin regulate chondrocyte metabo- lism? We will determine the effects of PGRN, Atsttrin, TNF? and TL1A on chondrocyte metabolism, their sig- naling pathways, target gene expression profiling and interplay in chondrocytes (SA#1A); whether PGRN and Atsttrin signaling and target gene expressions depends on 14-3-3? in chondrocytes (SA#1B); and whether the PGRN/Atsttrin regulates chondrocytes through modulating TL-1A/DR3 pathway as well (SA#1C). (2) Does PGRN/Atsttrin have therapeutic and protective role in OA, and what are the mechanisms of its action in OA? We will determine whether recombinant Atsttrin, similar to PGRN, reverses the susceptibility of PGRN-/- mice to OA challenge, and whether PGRN and Atsttrin ameliorate existing OA(SA#2A); whether chondrocyte- expressed 14-3-3? is important for PGRN/Atsttrin?s role in OA(SA#2B); and whether DR3 pathway also con- tributes to PGRN/Atsttrin?s protective role in OA (SA#2C). Completion of the proposed research will not only elucidate the importance of 14-3-3? and DR3 in PGRN-mediated regulation of chondrocytes and OA, but may also lead to the development of novel therapeutic strategies for treating OA and other degenerative diseases.